The central hypothesis of this research program is that age-related changes in the distribution of T cell subsets revealed by differences in P- glycoprotein (PGP) can account for an important proportion of the senescent decline in T cell function. Aging leads to an increase in the fraction of mouse T cells that can extrude the fluorochrome R123. This translocation is mediated by PGP, the ATP-dependent plasma membrane pump that mediates multiple drug resistance in tumor cells. R123 efflux delineates age sensitive subpopulations in each of the four conventionally accepted T cell subsets: R123hi T cells are gradually replaced by R123lo cells within the naive and memory pools of CD4 and CD8 cells. Within the CD4 memory subset, only R123hi T cells can respond to anti-CD3 and IL-2 by proliferation and il-4 secretion. In the current proposal, Aim #1 is to determine if R123hi and R123lo CD4 memory T cells differ in the spectrum of lymphokines produced in responses to two infectious agents (influenza virus and schistomes). Aim #2 is to determine whether R123hi and R123lo cells differ in their requirements for activation using antibodies to CD3 and costimulatory signals (CD4, CD28, and PMA). The results will shed light on the functional significance of the age-related shift in R123 subsets, an on the cellular basis for age-related changes in memory T cell responsiveness. These two aims together will show whether the poor responses of the R123lo T cells to anti-CD3 reflect a generalized anergy, a need for specific costimuli, an altered pattern of lymphokine production, or a preference for specific classes of cognate antigen, and will also show which aspects of age-dependent memory T cell dysfunction are attributable to R123 subset shifts and/or to changes within the R123-delineated subsets. Aim #3 is to see whether the poor proliferative responses of R123lo T cells to Il-2 reflect defects in Il-2R expression or response (or both), and whether similar defects are seen after stimulation with CD3/IL-2 independent stimuli such as PMA an Il-4. Aims #4 and #5 compare R123hi and R123lo CD4 T cells from young and old mice in two other measures of T cell help: ability to stimulate Ig production from B cells, and ability to give rise to clones of antigen-responsive memory T cells. The studies of Aim #6 will provide additional information about the biochemical bases for the increases of PGP in the R123lo subsets, including measures of PGP mRNA and PK-C mediated PGP phosphorylation. The results of these studies should provide a comprehensive picture of the effects of aging on the generation, longevity, and functional abilities of CD4 memory T cells, and test the idea that differences between R123hi and R123lo T cells contribute to these aspects of immunosenescence, while also providing insights into the underlying functional and biochemical differences between the R123hi and R123lo in young and old animals.